Cyclic peptides comprise a large and diverse group of secondary metabolites many of which are important as toxins and as antibiotics. Molecular genetic studies of cyclic peptide biosynthesis could lead to the genetic engineering of novel compounds with potential medical usefulness. HC-toxin is a cyclic tetrapeptide involved in the disease interaction between the producing fungus, Cochliobolus carbonum, and its host, maize, and is also cytostatic at nanomolar concentrations against cultured mammalian cancer cells. It contains an unusual epoxide-containing amino acid called Aeo which is required for activity. Four other, unrelated fungi are known to produce cyclic tetrapeptides containing Aeo. The production of HC-toxin is controlled by a single Mendelian locus called TOX2. We have recently cloned and sequenced a 22-kb region of DNA that is found only in isolates of the fungus that make HC-toxin. This region contains three genes, one of which is a 15.7-kb open reading frame (called HTS1) encoding a 570 kDa tetrapartite cyclic peptide synthetase called HC- toxin synthetase. This enzyme is the central enzyme in the assembly of HC- toxin, but does not synthesize Aeo. We propose to (1) study the structure of HC-toxin synthetase using biochemical and molecular genetic tools, in order to understand better the function of this kind of ecologically and pharmacologically-important enzyme, (2) study the biosynthesis of Aeo, relying on molecular genetics and the purification and identification of a novel Aeo precursor which accumulates when the genes for HC-toxin synthetase are mutated, (3) use the knowledge gained from our study of HTS1 and Aeo biosynthesis to isolate the corresponding genes from the four unrelated fungi that make closely related cyclic tetrapeptides. This will provide essential comparative information that will help us analyze the structure and function of all cyclic peptide synthetases, with the ultimate goal of biosynthesizing novel cyclic peptides. It will also allow us to test the hypothesis that HTS1 and TOX2 moved into C. carbonum from one of the other fungi (or vice versa) by horizontal gene transfer.